The present invention relates to methods of preparing biological material, for use in various experimental, diagnostic or therapeutic uses, including immunotherapy treatment or prophylaxy of tumors. More particularly, the present invention relates to methods of preparing membrane vesicles (in particular exosomes) released by various types of mammalian cells, comprising diafiltration and/or density cushion centrifugation. The invention also provides novel methods for characterizing and analysing exosome preparations, which can be used in quality control assay for the purpose of pharmaceutical product production. The invention is suitable to produce pharmaceutical grade preparations of such membrane vesicles and to fully characterize said preparations, for use in human beings.
Membrane vesicles are essentially spherical vesicles, generally less than 130 nm in diameter, composed of a lipid bilayer containing a cytosolic fraction. Particular membrane vesicles are more specifically produced by cells, from intracellular compartments through fusion with the plasmic membrane of a cell, resulting in their release in biological fluids or in the supernatant of cells in culture. Such vesicles are generally referred to as exosomes. Exosomes arc more particularly between about 30 and about 120 nm, preferably 50 and 90 nm, more specifically between about 60 and 80 nm in diameter and, advantageously, carry membrane proteins (particularly major histocompatibility complex proteins or other protein which directly or indirectly participate in antigen presentation). In addition, depending on their origin, exosomes comprise membrane proteins such as MHC I, MHC II, CD63, CD81 and/or HSP70 and have no endoplasmic reticulum or Golgi apparatus. Furthermore, exosomes are void of nucleic acids (e.g. DNA or RNA).
Exosome release has been demonstrated from different cell types in varied physiological contexts. In particular, it has been demonstrated that B lymphocytes release exosomes carrying class II major histocompatibility complex molecules, which play a role in antigenic presentation (Raposo et al., J. Exp. Med. 183 (1996) 1161). Similarly, it has been demonstrated that dendritic cells produce exosomes (i.e., dexosomes, Dex), with specific structural and functional characteristics and playing a role in immune response mediation, particularly in cytotoxic T lymphocyte stimulation (Zitvogel et al., Nature Medicine 4 (1998) 594). It has also been demonstrated that tumor cells secrete specific exosomes (i.e., texosomes, Tex) in a regulated manner, carrying tumor antigens and capable of presenting these antigens or transmitting them to antigen presenting cells (patent application No. WO99/03499). It is also known that mastocyte cells accumulate molecules in intracellular vesicular compartments, which may be secreted under the effect of signals (Smith and Weis, Immunology Today 17 (1996) 60). Therefore, as a general rule, cells appear to emit signals and communicate with each other via membrane vesicles that they release, which may carry antigenic proteins (or polypeptides or peptides), MHC molecules or any other signal (cytokine, growth factor, etc.) with specific structural and functional characteristics, produced in different physiological situations. These vesicles, particularly exosomes, thus represent a product of particular interest for diagnostic, vaccination or therapeutic applications or to deliver molecules of interest. Therefore, it would be of particular interest to have an effective method that could be used at an industrial scale to prepare membrane vesicles compatible with biological use, particularly pharmacological use.
Conventional methods to prepare membrane vesicles (e.g. exosomes) involve a series of differential centrifugation steps to separate the vesicles from cells or cell debris present in the culture medium. In this regard, the documents mentioned above essentially describe the preparation of vesicles with a series of centrifugations at 300 g, 10,000 g and 70,000 g or 100,000 g, upon which the resulting pellet at the bottom of the tube is resuspended to {fraction (1/1000)}th its original volume with a saline solution to constitute a concentrated exosome solution. However, these methods are essentially unsuitable for clinical applications for a number of reasons: 1) length of time, 2) scale-up and validation in GMP environment, 3) significant risk of contamination by cell debris, 4) poor reproducibility due to operator variability, 5) aggregation of exosomes resulting from pelleting (high localized exosome concentration in pellet) and 6) low recovery at end of processing. There is therefore a need for improved methods of preparing membrane vesicles, suitable with industrial constraints and allowing production of vesicle preparations of therapeutic quality.
International application nxc2x0 PCT/FR00/00105 discloses methods of preparing membrane vesicles through chromatographic techniques, such as anion exchange chromatography and/or gel permeation chromatography.
The present invention now provides novels methods of preparing membrane vesicles in high yields, high purity, and in relatively short periods of time. The present invention also discloses methods of characterizing (or analyzing or dosing) a membrane vesicle preparation, which can be used in pharmaceutical production to determine the activity, phenotype and/or quantity of vesicles. The invention now allows the production and characterization of clinically acceptable lots of membrane vesicles, with reproducibility, limited operator variation, and increased product quality. This invention further relates to methods of removing particulate bodies, such as haptoglobin, from various medium or compositions, the resulting compositions and media and their uses.
More specifically, an aspect of the present invention resides in methods of preparing membrane vesicles using density cushion centrifugation.
Another aspect of the present invention resides in methods of preparing membrane vesicles using a series of ultrafiltration steps and/or clarification step, more specifically a combination of a concentration and diafiltration by ultrafiltration, preferably preceded by a clarification.
Another aspect of this invention resides in methods of preparing membrane vesicles using a combination of density cushion centrifugation and ultrafiltration and/or clarification step, more specifically a combination of a concentration and diafiltration by ultrafiltration, preferably preceded by a clarification, followed by density cushion centrifugation.
In a particular aspect, the method of this invention comprises a density cushion centrifugation preceded or followed by a diafiltration.
The method of this invention can be applied to various biological samples containing membrane vesicles, including a biological fluid, a culture supernatant, a cell lysate or a pre-purified solution. In a particular embodiment, the method is used to prepare (e.g., purify or separate or isolate) membrane vesicles from a biological sample enriched with membrane vesicles.
A particular aspect of the present invention resides in a method of preparing membrane vesicles from a biological sample, comprising:
a. the culture of a population of membrane vesicle-producing cells under conditions allowing the release of the vesicles,
b. a membrane vesicle enrichment step, and
c. the treatment of said enriched biological sample by density cushion centrifugation.
In a further preferred embodiment, the membrane vesicle-producing cells are cultured in a culture medium with reduced particulate bodies"" content, preferably a medium deprived of haptoglobin aggregates. As will be demonstrated in this application, the use of such a medium allows increased production yields and/or higher purity and quality levels to be achieved.
The enrichment step may comprise one or several centrifugation, clarification, ultrafiltration, nanofiltration, affinity chromatography and/or diafiltration steps. More preferably, the enrichment step comprises a clarification and/or a concentration and/or a diafiltration.
The preparation of exosomes may be collected from step c) by any appropriate means, including pipetting or with a needle.
In a preferred embodiment, the method further comprises a sterile filtration d) of the preparation from step c.
The present invention can be used to prepare membrane vesicles from various origins, including membrane vesicles produced by antigen-presenting cells (such as macrophages, dendritic cells, B lymphocytes), tumor cells or any other cell or cell line producing vesicles, preferably transduced for antigens. It is particularly suited for preparing membrane vesicles produced by dendritic cells, preferably immature dendritic cells (i.e., dexosomes). Furthermore, the membrane vesicles or corresponding producing cells can be sensitized to one or several antigens, prior to, during or after preparation.
More preferred embodiments of this invention comprise:
a method of preparing membrane vesicles, comprising:
b. the culture of a population of antigen-presenting cells, in particular dendritic cells, under conditions allowing the release of membrane vesicles by antigen-presenting cells, in particular dendritic cells,
c. a membrane vesicle enrichment step, and
d. the isolation of the membrane vesicles using density cushion centrifugation.
a method of preparing membrane vesicles, comprising:
a. obtaining a population of immature dendritic cells
b. culturing the population of immature dendritic cells under conditions allowing the release of membrane vesicles by immature dendritic cells,
c. a membrane vesicle enrichment step, and
d. the isolation of the membrane vesicles using density cushion centrifugation.
As indicated above, an additional step of sensitization of the vesicles (or producing cells) to one or several particular antigens can be introduced in the process, either before step b), to sensitize the producing cells, or after step b), to sensitize directly the membrane vesicles.
a method of preparing membrane vesicles, comprising:
a. obtaining a population of antigen-presenting cells, more preferably immature dendritic cells,
b. sensitizing the antigen-presenting cells, more preferably the immature dendritic cells to one or several antigens,
c. culturing the population of antigen-presenting cells, more preferably immature dendritic cells under conditions allowing the release of membrane vesicles by antigen-presenting cells, more preferably immature dendritic cells,
d. a clarification of the culture supernatant,
e. a concentration of the clarified supernatant,
f. a diafiltration of the concentrated supernatant,
g. the isolation of the membrane vesicles using density cushion centrifugation, and
h. a sterile filtration of the membrane vesicles obtained in g.
Sterile filtration h) may be preceded by a buffer exchange step, for instance through diafiltration. A typical process scheme is depicted on FIG. 1.
Furthermore, the present invention also provides methods of removing particulate bodies from various media or compositions. More particularly, the invention demonstrates that conventional culture media contain particulate bodies, such as haptoglobin and related polymers (e.g., haptoglobin aggregates), and discloses methods of removing the same. More generally, the methods allow the production of culture media or any other biological products, such as blood proteins or polypeptides (or derivatives thereof), formulation solutions, fetal calf serum, etc., that are essentially deprived of haptoglobin aggregates.
Haptoglobin aggregates can exhibit immunosuppressive activity and thus affect the biological properties, safety and purity of membrane vesicles or other biological products, as will be further documented below. However, the issue of particular bodies was not addressed in the art, their presence in various biological products not determined, and efficient methods of removing the same not available. The invention now provides efficient methods to prepare high quality biological products, said resulting products also representing objects of this invention.
In this regard, the invention resides, generally, in a composition comprising a mammalian cell culture medium essentially free of particulate bodies, more preferably of haptoglobin aggregates.
The invention also resides in a cell culture medium deprived of haptoglobin aggregates.
The invention further resides in a composition of matter comprising antigen presenting cells (or any other membrane-vesicle producing cell, in particular dendritic cells) in a culture medium having a reduced particulate bodies content, more specifically that is essentially free of haptoglobin aggregates. More preferably, the culture medium is a culture medium treated to remove particulate or aggregate compounds, more particularly through ultrafiltration.
Another aspect of the present invention resides in method of producing or culturing antigen-presenting cells (or any membrane vesicle-producing cell), in particular dendritic cells, using culture or production media with reduced particulate bodies content. More preferably, the culture medium is a culture medium that is essentially free of haptoglobin aggregates, more specifically treated by ultrafiltration.
The invention is also suitable for the production of compositions of blood products that are essentially free of aggregated haptoglobin, as well as to the treatment of various buffer solutions prior to formulating products for pharmaceutical uses.
In this respect, the present invention also relates to a composition comprising a blood polypeptide or a derivative thereof, that is essentially deprived of haptoglobin aggregates. More particularly, this invention resides in a composition comprising a heat inactivated blood product that is essentially deprived of haptoglobin aggregates. Even more preferably, this invention relates to a composition of (heat inactivated) serum-albumin, more preferably human serum-albumin, essentially free of aggregated haptoglobin.
The invention also resides in a method of treating a biological product, more preferably a heat inactivated biological product, in order to reduce the amount of haptoglobin aggregates contained therein, comprising subjecting the product to filtration, more preferably ultrafiltration.
A particular object of this invention also resides in a method of preparing a biological product comprising (i) a heat inactivation of the biological product and (ii) a filtration of the heat inactivated biological product. More preferably, the method further comprises the step of (iii) concentrating the filtered, heat inactivated biological product and/or (iv) the conditioning thereof. The method can be used for various biological products including any protein or polypeptide (or derivatives thereof) isolated (or extracted) from mammalian biological fluids such as human blood or plasma or serum. As will be further documented in this application, this method allows, for the first time, the production of heat inactivated biological products having a reduced content in haptoglobin aggregates, more preferably essentially free of haptoglobin aggregates and thus with increased safety. The method is particularly suited for the preparation of pharmaceutical proteins extracted from blood or plasma such as serum-albumin, more preferably a human serum-albumin, gamma immunoglobulin, coagulation factors, etc.
The invention also resides in a method of treating a serum preparation, more preferably a fetal calf serum preparation, to reduce the amount of haptoglobin aggregates contained therein, comprising subjecting the preparation to filtration, more preferably ultrafiltration.
As will be further documented below, the expression xe2x80x9cessentially freexe2x80x9d indicates that the composition or medium contains less than about 1 ppm of haptoglobin aggregates, more preferably less than about 0.5 ppm, even more preferably no detectable haptoglobin aggregates by SDS PAGE analysis as well as by quantitative ELISA.
Another aspect of this invention resides in methods of analyzing or characterizing (or dosing) membrane vesicles in a preparation, in order to determine their phenotype and/or activity and/or quantity.
More particularly, an aspect of this invention lies in a method of characterizing membrane vesicles, comprising contacting the membrane vesicles in parallel with two or more antibodies specific for marker components of membrane vesicles and determining the formation of antigen-antibody immune complexes.
Another particular aspect comprises a method of characterizing the activity of a preparation of membrane vesicles, comprising contacting super-antigen-loaded vesicles with T cells in the presence of accessory cells, and determining the activation of the T cells.
The invention also provides a method of dosing membrane vesicles in a sample, comprising (i) loading the sample onto a solid support, (ii) contacting the support with an anti-class II antibody (or other relevant antibodies) and, (iii) determining the presence of antibody-antigen immune complexes.
The invention also comprises compositions comprising (i) membrane vesicles, (ii) a buffering agent aid (iii) a cryoprotectant or a stabilizing compound.
Other aspects of the present invention include kits, diagnostic assays, compositions of membrane vesicles, device(s) for preparing membrane vesicles, or antigen-loaded antigen-presenting cells (such as dendritic cells) or membrane vesicles.
The invention is particularly suited for preparing dexosomes (i.e., membrane vesicles produced by dendritic cells) or texosomes (i.e., membrane vesicles produced by tumor cells), more particularly of human origin. These membrane vesicles can be used in various experimental, biological, therapeutic, diagnostic or prophylactic applications. In particular, the membrane vesicles can be used to modulate an immune response in a subject, in particular in pathological conditions such as cancers, auto-immune diseases, allergy, asthma, inflammation and the like.